Biomarkers for inflammation of the liver

ABSTRACT

The invention relates to a method for the diagnostic investigation of biological samples from a person for inflammation of the liver, in particular hepatic fibrosis and/or cirrhosis of the liver, where the sample is investigated for one or more proteins as markers of inflammation of the liver, in particular hepatic fibrosis and/or cirrhosis of the liver, where a concentration of the proteins which is elevated or decreased by comparison with the healthy state indicates the presence of an inflammation of the liver, in particular a hepatic fibrosis and/or cirrhosis of the liver.; The proteins are selected from the group of ER6Q, vimentin, actin alpha 1 skeletal muscle protein, hMFAP 4, tropomyosin, PTGES 2, amyloid P component, transgelin, calponin 1, homo sapiens p20 protein, 17 kDa myosin light chain, H chain H Igg B12, prolyl 4-hydroxylase, beta subunit methylenetetrahydrofolate dehydrogenase 1, PRO2619, aldehyde dehydrogenase 1, fibrinogen alpha chain preproprotein, fructose-bisphosphate aldolase B, argininosuccinate synthetase, Eefla2, AT P 5 Al, alpha-2 actin, regucalcin, serum albumin, mitochondrial malate dehydrogenase, mitochondrial acetoacetyl-CoA thiolase or in each case a partial sequence thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 12/377,058, filed Jun. 26, 2009, which is a national stageapplication under 35 U.S.C. § 371 of PCT/DE2007/004127, filed Aug. 10,2007, which claims benefit of German application 102006037613.7, filedAug. 10, 2006 and German application 102006048249.2, filed Oct. 12,2006. The entire contents of each of these applications are herebyincorporated by reference herein.

DESCRIPTION

The invention relates to a method for the diagnostic study of biologicalsamples of a human for inflammation of the liver, in particular hepaticfibrosis and/or cirrhosis of the liver, the sample being studied for oneor more proteins as a marker for inflammation of the liver, inparticular hepatic fibrosis and/or cirrhosis of the liver, aconcentration of the proteins which is elevated or decreased in relationto the healthy state indicating the presence of an inflammation of theliver, in particular a hepatic fibrosis and/or cirrhosis of the liver.

Approximately 170 million humans are chronically infected with thehepatitis C virus (HCV) worldwide. The course of the illness variessignificantly between the patients; while approximately 20% of thepatients develop cirrhosis of the liver within 20 years, in otherpatients, a development of this type is not to be observed even afterstill longer periods of time. An array of factors may be identifiedwhich increase the probability of a hepatic fibrosis and/or cirrhosis ofthe liver, inter alia, male sex, alcohol misuse, co-infection with HIVor Schistosoma mansoni, genetic predisposition, and advanced age uponthe infection.

Above all, the hepatic stellate cells (HSC), which are responsible in anormal liver in the rest state for the storage of vitamin A inparticular, are responsible for the development of a liver fibrosisand/or cirrhosis of the liver. In contrast, in a fibrotic liver, theyare activated, proliferate, and develop into myofibroblastic cells.These myofibroblasts produce large quantities of collagen, down-regulatethe production of matrix metalloproteinases (MMP), and display anincreased expression of the physiological inhibitors of the MMP (TIMP).With increasing collagen accumulation, the fibrosis of the liverdevelops further, which finally may result in organ failure.

In particular peg-interferon alpha and ribavirin are used for theantiviral treatment of chronic hepatitis C. Although many patients maybe successfully treated in this way, the therapy remains unsuccessful inat least 50% of the patients, who are infected with the HCV genotype 1,which is the most widespread in the Western world. This is similarlytrue for patients who are infected with the HCV genotype 4, whichfrequently occurs in Egypt. In addition, the costs of permanentantiviral treatment are immense and the treatment is connected withsignificant side effects. In patients who are in a significantlyadvanced stage, the antiviral treatment again no longer results in thedesired success. Therefore, there is a need to be able to betterdiagnose a fibrosis in hepatitis patients and thus the occurrence ofcirrhosis of the liver, to be able to provide the treating physicianwith the capability of deciding whether an antiviral treatment isadvisable and promising.

An array of noninvasive markers has already been used in the past forthe detection of liver fibrosis, among them the so-called acti-test orfibro-test, pro-collagen III-peptide (PIIIP), hyaluronic acid, matrixmetalloproteinases (MMP) and their inhibitors (TIMP) (T. Poynard et al.,Expert Rev Mol Diagn. 2005, 5 (1): 15-21; V. Leroy et al., J Hep 2001,35 (1):26). However, all of these markers only show a limitedsensitivity and specificity, because of which there is a further demandfor more suitable biomarkers.

Proceeding from the described prior art, the object therefore presentsitself of providing an improved method for studying biological samplesfor inflammation of the liver and/or hepatic fibrosis and/or cirrhosisof the liver, in which novel markers are used.

The object is achieved according to the invention by a method forstudying biological samples of a human for inflammation of the liver, inparticular hepatic fibrosis and/or cirrhosis of the liver, the samplebeing studied for one or more proteins as a marker for an inflammationof the liver, in particular hepatic fibrosis and/or cirrhosis of theliver, and an elevated level of the proteins indicating the presence ofan inflammation of the liver, in particular hepatic fibrosis and/orcirrhosis of the liver, the proteins being selected from a groupcomprising: ER60, vimentin, actin alpha 1 skeletal muscle protein,hMFAP4, tropomyosin, PTGES2, amyloid-P-component, transgelin, calponin1, Homo sapiens p20 protein, 17 kDa myosin light chain, H chain H IgGB12, prolyl 4-hydroxylase, beta subunit.

Furthermore, the invention also relates to a corresponding method inwhich a decreased level of the proteins indicates the presence of aninflammation of the liver, in particular hepatic fibrosis and/orcirrhosis of the liver, in this case the proteins being selected from agroup: methylene tetrahydrofolate dehydrogenase 1, PRO2619, aldehydedehydrogenase 1, fibrinogen alpha-chain preproprotein,fructose-bisphosphate-aldolase B, argininosuccinate synthetase, EEF1A2,ATP5A1, alpha-2-actin, regucalcin, serum albumin, mitochondrial malatedehydrogenase, mitochondrial acetoacetyl-CoA thiolase.

Both for the up-regulating and also for the down-regulating proteins,the study may also be performed via the determination of partialsequences of the biomarkers (also: marker proteins) according to theinvention. In particular, such partial sequences preferably comprise 60%of the amino acid sequence of a biomarker according to the invention, inparticular 70% and more, 80% and more, in particular 90 to 95%.

In the context of this invention, the term inflammation of the livercomprises any form of hepatitis, but particularly hepatic fibrosis up tocirrhosis of the liver (on the terms, please see the relevantPschyrembel, Klinisches Wörterbuch [Clinical Dictionary], 260th edition,2004, Berlin, for example). Hepatic fibrosis and cirrhosis of the liverare preferred according to the invention.

Furthermore, the invention also relates to the diagnosis of inflammationof the liver, in particular hepatic fibrosis and/or cirrhosis of theliver, a determination of at least one protein selected from the groupcomprising: ER60, vimentin, actin alpha 1 skeletal muscle protein, hMFAP4, tropomyosin, PTGES 2, amyloid-P-component, transgelin, calponin 1,Homo sapiens p20 protein, 17 kDa myosin light chain, H chain H IgG B12,prolyl 4-hydroxylase, beta subunit, methylene tetrahydrofolatedehydrogenase 1, PRO2619, aldehyde dehydrogenase 1, fibrinogenalpha-chain preproprotein, fructose bisphosphate aldolase B,argininosuccinate synthetase, EEF1A2, ATP5A1, alpha-2-actin, regucalcin,serum albumin, mitochondrial malate dehydrogenase, mitochondrialacetoacetyl-CoA thiolase, or a partial sequence thereof in each casebeing performed on a patient to be studied.

Furthermore, a combination of such biomarkers and/or marker proteinsaccording to the invention is possible for the diagnosis according tothe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows pictures of proteins, separated using 2-D gelelectrophoresis, from fibrotic and non-fibrotic cells from cirrhoticliver parenchyma of seven patients who suffered from cirrhosis of theliver connected with hepatitis C.

FIG. 2 shows Western blot analysis of tropomyosin in sera of patentshaving cirrhosis of the liver.

The cited proteins may be identified as potential biomarkers during aproteome analysis of fibrotic tissue in comparison to non-fibrotictissue. For this purpose, liver biopsy samples were taken from patientsinfected with hepatitis C. The samples were homogenized with lysisbuffer in a manual homogenizer and freed of DNA and other cell materialto obtain a protein concentrate. The proteins were labeled using apigment and subjected to a 2-D polyacrylamide gel electrophoresis usingisoelectric focusing in the first dimension and SDS gel electrophoresisin the second dimension. The results were compared for fibrotic andnon-fibrotic cells with the aid of software suitable for this purpose,to detect and quantify the spots which were amplified or decreased inthe fibrotic sample in comparison to the non-fibrotic sample. Forexample, the ImageQuant™ software from GE Healthcare in connection withthe DeCyder software of the same firm may be executed as the software.The emission of the pigments, using which the proteins were labeled, wasmeasured and analyzed.

The further analysis was performed with the aid of LC-ESI-MS (liquidchromatography—electrospray ionization—mass spectrometry). Firstly theproteins were decomposed into individual peptide fragments with the aidof trypsin in the gel in which the samples had previously beenseparated. These fragments were separated from one another with the aidof reversed phase HPLC and studied using mass spectrometry to identifythe individual proteins. Of course, other suitable mass spectrometrymethods may also be applied for this purpose, such as MALDI-TOF-MS.

The following proteins were able to be identified in the studies, whichwere up-regulated (fold change positive) or down-regulated (fold changenegative) in fibrotic cells in relation to non-fibrotic cells:

NCBI accession Identified protein Fold change Up-regulated proteins:IPI00025252.1 ER60 protein 26.9 IPI00418471.5 vimentin 5.6 IPI00448938.1H chain H IgG B12 14.7 IPI00697648.1 actin alpha 1 skeletal muscleprotein 6.5 IPI00022792.3 hMFAP 4 45.1 IPI00455050.1 sarcomeretropomyosin kappa 87.1 IPI00014581.1 TPM1 human tropomyosin alpha-chain28.7 IPI00220709.3 beta tropomyosin 52.4 IPI00010779.3 tropomyosin 420.6 IPI00303568.3 PTGES 2 6.1 IPI00010796.1 prolyl 4-hydroxylase, betasubunit 4.6 IPI00022391.1 amyloid P-component, serum 7.3 IPI00216138.5transgelin 15 IPI00021264.1 calponin 1 21.1 IPI00022433.5 Homo sapiens p20 protein [pir B53814] 16.9 IPI00718271.2 17 kDa myosin light chain 8.4Down-regulated proteins: IPI00218342.9 methylene tetrahydrofolate −9.37dehydrogenase 1 IPI00745872.1 PRO2619 −3.2 IPI00218914.4 aldehydedehydrogenase 1 −7.7 IPI00029717.1 fibrinogen alpha-chain preprotein−6.4 IPI00218407.5 fructose bisphosphate aldolase B −16.6 IPI00020632.4argininosuccinate synthetase −11.0 IPI00014424.1 EEF1A2 −6.4IPI00440493.2 ATP5A1 −5.8 IPI00708487.1 alpha-2-actin; alpha cardiacactin −8.6 IPI00017551.1 regucalcin (senescence marker protein 30) −13.3IPI00708398.1 ABBOS-serum albumin precursor −13.3 IPI00291006.1mitochondrial malate dehydrogenase −5.9 precursor IPI00030363.1mitochondrial acetoacetyl-CoA thiolase −5.9 precursor NCBI: NationalCenter for Biotechnology Information

The sequence information for several of the identified proteins isspecified hereafter. The peptide sequences which, on the one hand,resulted in identification of the proteins and, on the other hand,permit a differentiation of the protein isoforms (compare also sequencelisting as the appendix to the patent application), have a darkunderlay.

Tropomyosin SEQ ID NO 1MDAIKKKMQMLKLDKENALDRAEQAEADKKAAEDRSKQLEEDIAAKEKLL SEQ ID NO 2MDAIKKKMQMLKLDKENALDRAEQAEADKKAAEDRSKQLEEDIAAKEKLL SEQ ID NO 3MDAIKKKMQMLKLDKENALDRAEQAEADKKQAEDRSKQLEEEQQALQKKL SEQ ID NO 4MDAIKKKMQMLKLDKENALDRAEQAEADKKAAEDRSKQLEDELVSLQKKL SEQ ID NO 5------------------------------------MAGLNSLEAVKRKI SEQ ID NO 1RVSEDERDRVLEELHKAEDSLLAAEEAAAKAEADVASLNRRIQLVEEELD SEQ ID NO 2RVSEDERDRVLEELHKAEDSLLAAEEAAAKAEADVASLNRRIQLVEEELD SEQ ID NO 3KGTEDEVEKYSESVKEAQEKLEQAEKKATDAEADVASLNRRIQLVEEELD SEQ ID NO 4KGTEDELDKYSEALKDAQEKLELAEKKATDAEADVASLNRRIQLVEEELD SEQ ID NO 5QALQQQADEAEDRAQGLQRELDGERERREKAEGDVAALNRRIQLFEEELD SEQ ID NO 1RAQERLATALQKLEEAEKAADESERGMKVIESRAQKDEEKMEIQEIQLKE SEQ ID NO 2RAQERLATALQKLEEAEKAADESERGMKVIESRAQKDEEKMEIQEIQLKE SEQ ID NO 3RAQERLATALQKLEEAEKAADESERGMKVIENRAMKDEEKMELQEMQLKE SEQ ID NO 4RAQERLATALQKLEEAEKAADESERGMKVIESRAQKDEEKMEIQEIQLKE SEQ ID NO 5RAQERLATALQKLEEAEKAADESERGMKVIENRAMKDEEKMEIQEMQLKE SEQ ID NO 1AKHIAEDADRKYEEVARKLVIIESDLERAEERAELSEGKCAELEEELKTV SEQ ID NO 2AKHIAEDADRKYEEVARKLVIIESDLERAEERAELSEGKCAELEEELKTV SEQ ID NO 3AKHIAEDSDRKYEEVARKLVILEGELERSEERAEVAESRARQLEEELRTM SEQ ID NO 4AKHIAEDADRKYEEVARKLVIIESDLERAEERAELSEGQVRQLEEQLRIM SEQ ID NO 5AKHIAEEADRKYEEVARKLVILEGELERAEERAEVSELKCGDLEEELKNV SEQ ID NO 1TNDLKSLEAQAEKYSQKEDRYEEEIKVLSDKLKEAETRAEFAERSVTKLE SEQ ID NO 2TNDLKSLEAQAEKYSQKEDRYEEEIKVLSDKLKEAETRAEFAERSVTKLE SEQ ID NO 3DQALKSLMASEEEYSTKEDKYEEEIKLLEEKLKEAETRAEFAERSVAKLE SEQ ID NO 4DQTLKALMAAEDKYSQKEDRYEEEIKVLSDKLKEAETRAEFAERSVTKLE SEQ ID NO 5TNNLKSLEAASEKYSEKEDKYEEEIKLLSDKLKEAETRAEFAERTVAKLE SEQ ID NO 1KSIDDLEDELYAQKLKYKAISEELDHALNDMTSI SEQ ID NO 2KSIDDLEDELYAQKLKYKAISEELDHALNDMTSI SEQ ID NO 3KTIDDLEETLASAKEENVEIHQTLDQTLLELNNL SEQ ID NO 4KSIDDLEEKVAHAKEENLSMHQMLDQTLLELNNM SEQ ID NO 5KTIDDLEEKLAQAKEENVGLHQTLDQTLNELNCI Transgelin SEQ ID NO 6MANKGPSYGMSREVQSKIEKKYDEELEERLVEWIIVQCGPDVGRPDRGRL SEQ ID NO 7MANKGPSYGMSREVQSKIEKKYDEELEERLVEWIIVQRGPDVGRPDRGRL SEQ ID NO 6GFQVWLKNGVILSKLVNSLYPDGSKPVKVPENPPSMVFKQMEQVAQFLKA SEQ ID NO 7GFQVWLKNGVILSKLVNSLYPDGSKPVKVPENPPSMVFKQMEQVAQFLKA SEQ ID NO 6AEDYGVIKTDMFQTVDLFEGKDMAAVQRTLMALGSLAVTKNDGHYRGDPN SEQ ID NO 7AEDYGVIKTDMFQTVDLFEGKDMAAVQRTLMALGSLAVTKNDGHYRGDPN SEQ ID NO 6WFMKKAQEHKREFTESQLQEGKHVIGLQMGSNRGASQAGMTGYGRPRQII SEQ ID NO 7WFMKKAQEHKRSFTESQLQEGKHVIGLQMGSNRG---------------- SEQ ID NO 6 SSEQ ID NO 7 -SEQ ID NO 1: sarcomere tropomyosin kappa, TPM1-kappa; NCBI accession:IPI00455050.1SEQ ID NO 2: sarcomere tropomyosin kappa; NCBI accession: IPI00455050.1SEQ ID NO 3: beta tropomyosin; NCBI accession: IPI00220709.3SEQ ID NO 4: TPM1 human tropomyosin 1 alpha-chain; NCBI accession:IPI00014581.1 SEQ ID NO 5: tropomyosin 4; NCBI accession: IPI00010779.3SEQ ID NO 6: transgelin; NCBI accession: IPI00216138.5SEQ ID NO 7: transgelin variant; NCBI accession: IPI00216138.5

FIG. 1 shows pictures of the proteins separated using 2-D gelelectrophoresis, fibrotic and non-fibrotic cells from cirrhotic liverparenchyma of a total of 7 patients, who suffered from cirrhosis of theliver connected with hepatitis C, having been used. The circular markingshows tropomyosin (beta), and the oval marking shows hMFAP 4. Thesimilarity of the pictures underlines the reproducibility of theresults.

In particular, tropomyosin, transgelin, calponin, hMFAP4, and vimentinhave been shown to the promising and preferred biomarkers according tothe invention. Furthermore, the tropomyosin may be sarcomere tropomyosinkappa, beta tropomyosin, TPM1 human tropomyosin, or tropomyosin 4.

The hMFAP4 cited is the human microfibrillar associated protein 4. Inaddition to the above-mentioned up-regulated proteins, however,down-regulated metabolic enzymes may also be used as a marker, inparticular argininosuccinate synthetase, methylene tetrahydrofolatedehydrogenase, fructose-1,6-biophosphate aldolase, mitochondrial malatedehydrogenase, and mitochondrial acetoacetyl-CoA thiolase.

Some of the identified proteins are discussed briefly hereafter, theexplanations provided therein solely being attempts at clarificationswhich are not to be understood as restrictive in any way in regard tothe object for which a patent is sought.

Regucalcin, also known as senescence marker protein-30 (SMP-30), plays asignificant role in maintaining the intracellular Ca2+level byactivation of Ca2+enzymes in the plasma membrane, the microsomes, andthe mitochondria. Regucalcin was down-regulated 13-fold in fibroticliver cells in comparison to healthy liver cells. This may indicate thatthe compensatory effects of regucalcin were decreased with respect tooxidative stress in diseased liver tissue.

Mitochondrial malate dehydrogenase and mitochondrial acetoacetyltransferase were both down-regulated by 5.9-fold in the fibrotic livertissue. It was possible to show that the interaction between hepatitis Cvirus core protein and the proteins NS3 and NS5 with mitochondriaresulted in the formation of reactive oxygen species (ROS), which may bean explanation of the weaker expression of the cited enzymes in fibroticcells.

A further indication of disturbed mitochondrial enzymes in fibroticliver cells is the decreased expression of the ATP synthasealpha-subunit (ATP5A1), which catalyzes ATP synthesis during oxidativephosphorylation.

Further effects of the hepatitis C virus core protein and the NS5Aprotein are the association with membranes of the endoplasmaticreticulum, the Golgi apparatus, and an amplification of theintracellular lipid accumulation by interaction with apolipoprotein Al(apoAl) or A2 (apoA2). The lipid transfer protein is correspondinglyinhibited or the synthesis of VLDL (very low density lipoproteins) isdisturbed. A significant down-regulation of theacetyl-CoA-acetyltransferase was able to be observed in fibrotic livercells (fold change: −5.88).

Metabolic disturbances in fibrotic livers also manifest themselves in adecreased expression of glycolytic enzymes such asfructose-1,6-bisphosphate aldolase (fold change: −16.6), enolase-1(2-phosphodiglycerate hydrolase), or glyoxylase.

Because of these metabolic dysfunctions, the protein synthesis ispartially significantly reduced in fibrotic liver cells: serum albumin(fold change: −13.2), which functions as a carrier for fatty acids,steroids, and thyroid hormones and stabilizes the extracellular liquidvolume, was only still expressed in a reduced way.

The process of liver regeneration in fibrotic liver cells is apparentlyimpaired by a weaker expression of methylene tetrahydrofolatedehydrogenase (fold change: −9.4), which catalyzes three sequentialreactions in the conversion of C-1 derivatives of tetrahydrofolate. Anon-reduced enzymatic function is significant for the normal cellularfunction, growth, and dedifferentiation.

Overall, a disturbance of the cellular equilibrium, the glycolyticreaction pathways, and the lipid compartmentalization and the metabolismappear to exist in fibrotic cells, which encourages the production ofreactive organic species (ROS). These in turn induce the synthesis ofTGF-β1 in hepatocytes and hepatic stellate cells, the strongestpromoters of hepatic fibrogenesis.

The proteins actin alpha (fold change: 6.5) and actin gamma (foldchange: 9.1) were able to be identified in the area of the amplifieddetectable proteins. These are the main components of the thin filamentsof muscle cells and the cytoskeleton of non-muscular cells. Actin isapparently a product of the HSC cells and occurs in an amplified way inthe event of hepatitis C-induced fibrosis.

A further identified protein is vimentin (fold change: 4.6). This ispresumed to be a product of the hepatic stellate cells from themesenchyma.

Various tropomyosin isoforms, which are apparently not produced byhepatocytes but by myofibroblastic hepatic stellate cells, partiallyhave the highest rates of amplification (fold change: 9.7 to 83.1).

The 34 kDa protein calponin is normally also expressed specifically insmooth muscle cells and binds calmodulin, actin, and tropomyosin. Inview of the myofibroblastic activated hepatic stellate cells, calponinwas also up-regulated in fibrotic cells according to the results of theproteome analysis (fold change: 18.5).

Transgelin (fold change: 15) is also presumed to be a product of hepaticstellate cells. Transgelin is a 22 kDa protein, which is also referredto as SM22-alpha and has structural similarities to calponin. Theamyloid component P, a glycoprotein which is composed of a pair ofnon-covalently bound pentamers, the subunit having a size from 23 to 25kDa, was also more strongly expressed in the event of fibrosis than inhealthy tissue (fold change: 7.3). The physiological function in hepaticfibrogenesis is unknown up to this point, but the overexpressionindicates an abnormal cellular process.

In summary, it may be stated that the overexpression or underexpressionof the proteins usable as biomarkers is to be attributed to a disturbedcellular equilibrium, impaired mitochondrial and metabolic enzymes,reduced cellular synthesis, and an amplified expression of cytoskeletalproteins during the process of apoptosis in fibrotic liver cells.

In the study of a sample for the proteins used as the biomarkers, asdescribed above, a procedure may be used in which, with the aid of a 2-Dgel electrophoresis, comprising isoelectrical focusing in the firstdimension and gel electrophoresis in the second dimension, a separationof the proteins is performed and the overexpression and/orunderexpression of specific proteins is proven by comparison of theprotein pattern to a non-fibrotic control sample. The gelelectrophoresis is preferably an SDS polyacrylamide gel electrophoresis.Corresponding software for analyzing the gels is obtainable, forexample, from GE Healthcare in the form of the DeCyder software.

To detect the proteins, the samples are preferably labeled using apigment before performance of the 2-D gel electrophoresis. The pigmentsare preferably fluorescent pigments. The use of Cy2, Cy3, and/or Cy5 isespecially preferred. These pigments are obtainable, for example, fromGE Healthcare, Freiburg, Germany. These are carbomethylindocyaninepigments, two indole molecules being connected via a carbon chain havingconjugated double bonds. The corresponding functionalized pigments maybe caused to react with the thiol groups of the side chains of thecysteine to link the proteins covalently to the pigments. For thepurpose of reduction of disulfide bridges, the sample is first reducedwith the aid of a suitable reducing agent, for example, with the aid oftris (2-carboxyethyl) phosphine hydrochloride (TCEP). Subsequently, areaction is performed with corresponding functionalized pigment untilfinally the reaction is stopped by adding DTT. However, thefunctionalization of other amino acid residues with the aid of thepigments is also conceivable, for example, the side chain of lysine.

The use of the Cy3, Cy5 pigment system is particularly advantageous inthat, in addition to the actual sample, an internal standard may also beused during the 2-D gel electrophoresis, the actual sample and theinternal standard being provided with different pigments (Cy3 and Cy5,respectively).

Of course, in addition to the cited pigments, other (fluorescent)pigments are also usable, which are known from the prior art, such asfluorescein or tetramethyl rhodamine.

The detection and the quantification of the proteins used as the markersmay also be performed with the aid of further protein diagnostic methodsknown to those skilled in the art, in particular employing radioactiveor fluorescence-marked antibodies. In particular, bioanalytical methodssuitable for this purpose are to be cited here, such asimmunohistochemistry, antibody arrays, luminex, ELISA,immunofluorescence, and radio immunoassays. The detection and thequantification of the proteins used as the markers may also be performedusing further bioanalytical methods suitable for this purpose, such asmass-spectrometry methods, e.g., MRM (multi-reaction monitoring) or AQUA(absolute quantification), with the aid of which the marker proteins maybe quantitatively measured.

The sample used for detecting the proteins may be a sample of livertissue which was removed with the aid of a biopsy. However, the use of(whole) blood, serum, or plasma samples, which are obviously easier toobtain, is also possible.

In addition to the described method, the invention also relates to theuse of the cited proteins as biomarkers for the detection of aninflammation of the liver, in particular of a hepatic fibrosis.

The cited marker proteins according to the invention may also be used infurther embodiments for differential diagnosis, in particulardifferential-diagnostic early recognition, course prognosis of the liverillness, judgment of the degree of severity, course judgmentaccompanying the treatment, etiology, and in vitro diagnostics.

In a further embodiment, the invention relates to a kit or diagnosticdevice for performing the method according to the invention, the kitcontaining at least one biomarker according to the invention (also:marker protein) together with detection reagents and further aids.

The following examples are used to explain the invention withoutrestricting the invention to these examples.

EXAMPLE Example 1 Sample Analysis

Liver parenchyma was taken from a total of seven patients having ahepatitis C infection, genotype 1, who had been subjected to a livertransplantation, immediately cooled on ice, and stored at −86° C. Sampletissues were separated between fibrotic tissue and healthy sectionsunder the microscope. The fibrotic material was taken along the fibroticseptum. The layers for the 2-D gel electrophoresis were stained usinghematoxylin and stored at −20° C. The isolated cells from themicrodissection were placed in 100 pL lysis buffer (tris HCl 30 mM;thiourea 2 M; urea 7 M, CHAPS 4%, pH 8.0) and subsequently broken up byapplying ultrasound (6×10 s pulses).

DNA and other cell residues were removed by a centrifugation (12,000 gfor five minutes). The protein concentration of the lysate wasascertained.

To prepare an internal standard, 3 mcg of the tissue lysate was reducedby application of 2 nmol tris-(2-carboxyethyl)-phosphine hydrochloride(TCEP) at 37° C. in the dark over a time span of one hour. Cy pigments(GE Healthcare, Freiburg, Germany) were diluted using nonaqueous DMFp.a. (2 nmol/pl) and 4 nmol Cy3 was admixed to the samples reduced withthe aid of TCEP. After an incubation time of 30 minutes at 37° C., thelabeling reaction was stopped by adding 4 pL DTT (1.08 g/mL).

The cysteine amino acids of the proteins to be studied (3500 fibroticcells, 2500 non-fibrotic cells) were also reduced by incubation with 2nmol TCEP at 37° C. in the dark for one hour, before 4 nmol Cy5 wasadded. After thorough mixing, the samples were reacted for 30 minutes at37° C. in the dark. The reaction was stopped by adding 10 μL DTT.

To prepare the isoelectric focusing, 10 μL ampholine 2-4 (GE Healthcare)was added. The Cy3-marked and Cy5-marked cells were concurrentlyprocessed further after thorough mixing.

Subsequently, a 2-D gel electrophoresis was performed, a 21.25 h voltagegradient being used for the isoelectric focusing. 125 mM tris, 40% (w/v)glycerol, 3% (w/v) SDS, 65 mM DTT, pH 6.8 was used for 10 minutes as anequilibration buffer. Subsequently, a polyacrylamide gel electrophoresiswas performed in the second dimension.

With the aid of a suitable scanner (Typhoon 9400, GE Healthcare), imageswere recorded after completed gel electrophoresis and analyzed with theaid of the ImageQuant software and the DeCyder software (GE Healthcare).In this way, a differential analysis was able to be performed in the gel(DIA), to detect and quantify the individual spots.

The proteins were cleaved at 37° C. overnight with the aid of the enzymetrypsin in 10 mM ammonium bicarbonate buffer (pH 7.8). The fragmentsgenerated in this way were extracted twice using an acetonitrile-formicacid mixture for further partitioning and mass-spectrometry study. Thiswas performed with the aid of online-RP-capillary HPLC, coupled withnano-ESI-MS (electrospray ionization mass spectrometry). Completeidentification of the protein spots was able to be performed with theaid of the HPLC-MS coupling and employing suitable protein databanks(NCBI, National Center for Biotechnology Information).

Example 2 Protein Tropomyosin and its Detection in Patient Sera

A protein detection using Western blot analysis was performed for thispurpose. The tropomyosin protein was able to be identified reproduciblyin patient sera of cirrhosis of the liver patients of various origins(FIG. 2).

FIG. 2: tropomyosin Western blot of the following patient sera:

1: (patient 1) hepatitis C (HepC) cirrhosis CHILD C

2: (patient 2) HepC cirrhosis CHILD C

3: (patient 3) HepC cirrhosis CHILD C

4: (patient 4) HepC cirrhosis CHILD C

5: (patient 5) HepC cirrhosis CHILD C

6: (patient 6) HepC cirrhosis CHILD A

7: (patient 7) HepC cirrhosis CHILD A

8: (patient 8) normal control

9: (patient 9) normal control

10: (patient 10) normal control

11: (patient 11) normal control

12: (patient 12) ethyl-toxic cirrhosis CHILD C

13: (patient 13) ethyl-toxic cirrhosis CHILD C

14: (patient 14) HepB fibrosis

15: (patient 15) HepC fibrosis

16: tropomyosin 0,005 μg

The patients were, on the one hand, patients infected with hepatitis Chaving cirrhosis of the liver and/or fibrosis, and, on the other hand,patients having ethyl-toxic cirrhosis of the liver and one hepatitis Bpatient having liver fibrosis. Patients having cirrhosis of the liverwere divided further into various CHILD classes, which provideinformation about the degree of severity of the cirrhosis, byincorporating various blood parameters. The classification is performedfrom CHILD A having a survival rate of approximately 100% for the nextyear up to CHILD C having a survival rate of approximately 30%. Atropomyosin band was not able to be detected in patient sera of healthynormal controls (FIG. 2, bands 8-11). Interestingly, tropomyosin wasable to be detected in hepatitis C patients having a CHILD Cclassification (FIG. 2, bands 1-5), while no protein band was detectablein cirrhotics having a CHILD A cirrhosis (FIG. 2, bands 6-7). Inaddition, ethyl-toxic cirrhosis of the liver were studied (CHILD Cclassification), which also showed a signal for tropomyosin inattenuated form and had a CHILD C classification (FIG. 2, bands 12-13).Furthermore, a tropomyosin band was able to be detected in the hepatitisB fibrosis patients, which was not possible in the hepatitis C fibrosispatients.

1. A kit or diagnostic device containing at least one marker protein together with detection reagents and further aids, wherein said at least one marker protein is selected from the group consisting of ER60, vimentin, actin alpha 1 skeletal muscle protein, hMFAP 4, tropomyosin, PTGES 2, amyloid P-component, transgelin, calponin 1, Homo sapiens p20 protein, 17 kDa myosin light chain, H chain H IgG B12, prolyl 4-hydroxylase beta subunit, methylene tetrahydrofolate dehydrogenase 1, PRO2619, aldehyde dehydrogenase 1, fibrinogen alpha-chain preproprotein, fructose-bisphosphate-aldolase B, argininosuccinate synthetase, EEF1A2, ATP5A1, alpha-2-actin, regucalcin, serum albumin, mitochondrial malate dehydrogenase, and mitochondrial acetoacetyl-CoA thiolase, or a partial sequence thereof in each case.
 2. A method for the diagnosis of inflammation of the liver, comprising the step of determining the concentration of at least one protein selected from the group consisting of ER60, vimentin, actin alpha 1 skeletal muscle protein, hMFAP 4, tropomyosin, PTGES 2, amyloid P-component, transgelin, calponin 1, Homo sapiens p20 protein, 17 kDa myosin light chain, H chain H IgG B12, prolyl 4-hydroxylase beta subunit, methylene tetrahydrofolate dehydrogenase 1, PRO2619, aldehyde dehydrogenase 1, fibrinogen alpha-chain preproprotein, fructose-bisphosphate-aldolase B, argininosuccinate synthetase, EEF1A2, ATP5A1, alpha-2-actin, regucalcin, serum albumin, mitochondrial malate dehydrogenase, and mitochondrial acetoacetyl-CoA thiolase, or a partial sequence thereof in each case, in a sample from a human patient.
 3. The method of claim 2, wherein said inflammation of the liver is hepatic fibrosis or cirrhosis of the liver.
 4. The method of claim 2, comprising the steps of determining the concentration of one or more proteins in a sample from a human, as markers for inflammation of the liver, and comparing the concentration of said one or more proteins in said sample with the amount of said one or more proteins in the healthy human state, wherein a concentration of said one or more proteins in said sample which is elevated in relation to the healthy state indicating the presence of an inflammation of the liver, and wherein said one or more proteins are selected from the group consisting of ER60, vimentin, actin alpha 1 skeletal muscle protein, hMFAP 4, tropomyosin, PTGES 2, amyloid P-component, transgelin, calponin 1, Homo sapiens p20 protein, 17 kDa myosin light chain, H chain H IgG B12, and prolyl 4-hydroxylase beta subunit, or a partial sequence thereof in each case.
 5. The method of claim 2, comprising the steps of determining the concentration of one or more proteins in a sample from a human, as markers for inflammation of the liver, and comparing the concentration of said one or more proteins in said sample with the amount of said one or more proteins in the healthy human state, wherein a concentration of said one or more proteins in said sample which is decreased in relation to the healthy state indicating the presence of an inflammation of the liver, and wherein said one or more proteins are selected from the group consisting of methylene tetrahydrofolate dehydrogenase 1, PRO2619, aldehyde dehydrogenase 1, fibrinogen alpha-chain preproprotein, fructose-bisphosphate-aldolase B, argininosuccinate synthetase, EEF1A2, ATP5A1, alpha-2-actin, regucalcin, serum albumin, mitochondrial malate dehydrogenase, and mitochondrial acetoacetyl-CoA thiolase, or a partial sequence thereof in each case.
 6. The method of claim 5, wherein said inflammation of the liver is hepatic fibrosis.
 7. The method according to claim 2, wherein the tropomyosin is sarcomere tropomyosin kappa, beta tropomyosin, TPM 1 human tropomyosin, or tropomyosin
 4. 8. The method according to claim 2, wherein determining the concentration of said one or more proteins is performed by immunohistochemistry, antibody arrays, luminex, ELISA, immunofluorescence, or radio immunoassays.
 9. The method according to claim 2, wherein determining the concentration of said one or more proteins is performed using mass-spectrometry methods.
 10. The method according to claim 2, wherein determining the concentration of said one or more proteins is performed by 2-D electrophoresis, an isoelectric focusing being performed in the first dimension, and a gel electrophoresis being performed in the second dimension.
 11. The method according to claim 10, wherein the gel electrophoresis is an SDS-polyacrylamide gel electrophoresis.
 12. The method according to claim 10, wherein the sample is labeled using a pigment before performing the 2-D gel electrophoresis.
 13. The method according to claim 12, wherein the pigment is Cy2, Cy3, and/or Cy5.
 14. The method according to claim 2, wherein the sample is a liver biopsy sample.
 15. The method according claim 2, wherein the sample is blood serum, blood plasma, or (whole) blood. 